How cells progress from stem cells to end-stage differentiation is poorly understood. Substantial evidence indicates that transcriptional regulator networks play a key role in this process. One possibility is that the network regulating differentiation is comprised of a series of switch-like steps that open certain options and preclude others. These switch-like processes emerge from properties of the dynamics of gene regulatory networks. An alternative, not mutually exclusive, hypothesis is that the path to differentiation is a series of incremental steps, each one modifying the output of the gene regulatory network. The latter hypothesis encompasses more tolerance for overlapping differentiation pathways as well as a greater degree of reversibility. These alternative hypotheses will be tested in the tractable model of the Arabidopsis root. The simplicity of its organization, with an accessible stem cell center that gives rise to all the other cells, makes it n ideal model in which to perturb and characterize the differentiation pathway in the context of a developing organ. Regulatory networks that control the asymmetric division of a stem cell population in the root and determine critical steps along the differentiation pathway have been characterized. A mathematical model has been derived to simulate network activity during the asymmetric cell division. Innovative approaches that will be used include the experimental determination of model parameters using fluorescence correlation spectroscopy and monitoring dynamics of network components employing 2-photon light sheet microscopy. Newly identified targets and upstream regulators will be added to the current networks. The design principles of the networks will be tested by targeted perturbations, examining regulators in new cellular contexts and through use of synthetic transcriptional regulators. Knowledge of the process by which the progeny of stem cells become differentiated will be critical to harnessing the power of induced pluripotent stem cells for therapeutic purposes such as tissue regeneration and for providing insight into the dedifferentiated state of tumor cells.